ORIGINAL RESEARCH
published: 29 June 2016
doi: 10.3389/fpls.2016.00943
An Efficient Method for Adventitious
Root Induction from Stem Segments
of Brassica Species
Sandhya Srikanth, Tsui Wei Choong, An Yan, Jie He and Zhong Chen *
Natural Sciences and Science Education, National Institute of Education, Nanyang Technological University, Singapore,
Singapore
Edited by:
Naser A. Anjum,
University of Aveiro, Portugal
Reviewed by:
Abu Hena Mostafa Kamal,
University of Texas at Arlington, USA
Appakan Shajahan,
Jamal Mohamed College, India
*Correspondence:
Zhong Chen
[email protected]
Specialty section:
This article was submitted to
Crop Science and Horticulture,
a section of the journal
Frontiers in Plant Science
Received: 22 December 2015
Accepted: 13 June 2016
Published: 29 June 2016
Citation:
Srikanth S, Choong TW, Yan A, He J
and Chen Z (2016) An Efficient
Method for Adventitious Root
Induction from Stem Segments
of Brassica Species.
Front. Plant Sci. 7:943.
doi: 10.3389/fpls.2016.00943
Plant propagation via in vitro culture is a very laborious and time-consuming process.
The growth cycle of some of the crop species is slow even in the field and the
consistent commercial production is hard to maintain. Enhanced methods of reduced
cost, materials and labor significantly impact the research and commercial production
of field crops. In our studies, stem-segment explants of Brassica species were found
to generate adventitious roots (AR) in aeroponic systems in less than a week. As such,
the efficiency of rooting from stem explants of six cultivar varieties of Brassica spp was
tested without using any plant hormones. New roots and shoots were developed from
Brassica alboglabra (Kai Lan), B. oleracea var. acephala (purple kale), B. rapa L. ssp.
chinensis L (Pai Tsai, Nai Bai C, and Nai Bai T) explants after 3 to 5 days of growing under
20 ± 2◦ C cool root zone temperature (C-RZT) and 4 to 7 days in 30 ± 2◦ C ambient
root zone temperature (A-RZT). At the base of cut end, anticlinal and periclinal divisions
of the cambial cells resulted in secondary xylem toward pith and secondary phloem
toward cortex. The continuing mitotic activity of phloem parenchyma cells led to a ring
of conspicuous white callus. Root initials formed from the callus which in turn developed
into ARs. However, B. rapa var. nipposinica (Mizuna) explants were only able to root in
C-RZT. All rooted explants were able to develop into whole plants, with higher biomass
obtained from plants that grown in C-RZT. Moreover, explants from both RZTs produced
higher biomass than plants grown from seeds (control plants). Rooting efficiency was
affected by RZTs and explant cuttings of donor plants. Photosynthetic CO2 assimilation
rate (Asat ) and stomatal conductance (gssat ) were significantly differentiated between
plants derived from seeds and explants at both RZTs. All plants in A-RZT had highest
transpiration rates.
Keywords: aeroponics, Brassica, explants, root zone temperature, rooting, stem-segment
INTRODUCTION
Green leafy vegetables, known for their high nutritional content, are consumed by humans
for good health and dietary benefits. The Brassicaceae family encompasses the Brassiceae tribe,
which includes wide varieties of agriculturally and economically significant species. The genus
Brassica, including kales, cabbages, broccoli, cauliflower, brussel sprouts, and kohlrabi, comprise
of biennially herbaceous plants classified by their characteristic morphology of edible parts
Frontiers in Plant Science | www.frontiersin.org
1
June 2016 | Volume 7 | Article 943
Srikanth et al.
Brassica Root Regeneration in Aeroponics
(Parkin et al., 2014). B. alboglabra, also known as Chinese kale
or Kai Lan, is among the ten most marketable vegetables in
Southeast Asian countries, such as Hong Kong, Thailand and
China (Rakow, 2004). B. oleracea var. acephala (purple curly
kale), related to the common cabbage, is a biennial temperate
crop that is cultivated as an annual. Their flower buds and leaves
are used as potherbs or greens (Velasco et al., 2007). Mizuna, a
cultivated variety of B. rapa var. nipposinica, is a leafy vegetable
commonly found in Japanese salads. Pai Tsai and Nai Bai are
the cultivated varieties of B. rapa L. ssp. chinensis that is gaining
popularity in Western menus where they are often steamed or stir
fried (Rochfort et al., 2006). Brassica species play a significant role
in agriculture and horticulture fields and contribute significantly
to economies and population health worldwide (Zhao, 2007).
Additionally, these Brassica species also represent an excellent
system for studying numerous aspects of plant biology (Cheng
et al., 2011).
Seed companies and industries face many challenges to
acquire seeds from Brassica cultivars as it takes an especially long
time to obtain. Conventional methods of hybrid seed production
involve selling inbred lines for at least ten generations, while
the development of homozygous plants of anther culture takes
at least a year (Yang et al., 1992). Further, unlike other crops,
Brassica seed production is limited to the fields with a minimum
5-year gap between seed crops and at least 2 years exclusion of
any Brassica species in the same field (Ellis, 2007). Hence, a quick
alternative in the commercial field would be to use vegetative
propagation via stem cuttings. This minimizes seed usage while
allowing for new plants to be developed within a week shorter
time interval.
Vegetative propagations, such as leaves, cladode, stem
or branch cuttings, are well-known methods of asexual
propagations. In general, stem cutting is the most popular
method of propagation for commercial plantings worldwide.
However, operating costs are high as a continuous supply of
fresh materials, such as peat moss, vermiculite, coir pith, root
trainers, and fungicides, are required for the existing stem cutting
propagation method. Rooting hormones, such as auxin, are
also required for de novo root formation in explants. Though
auxin stimulates root initiation, it also habitually leads to callus
formation and expression of genes that are not necessarily related
to root initiation (Welander et al., 2014). Since ARs may originate
independently and directly from explant tissue rather than from
callus (Fink, 1999), a more efficient rooting method with little or
no callus formation would be desirable. Thus, a new method that
could increase the speed of propagation whilst lower propagation
costs would be an ideal alternative approach.
Soilless culture systems are useful for both research and
commercial applications for food crops. An example is an
aeroponic system which allows for plants to grow whilst their
roots are suspended in air. As previous studies (Weathers and
Giles, 1988; Zobel, 1989; Luo et al., 2012) have suggested that
aeroponics is the optimum system for growing intact plants
or excised roots and tissue cultures, this research explores
the possibility of vegetative propagation of temperate Brassica
species (Asian greens) in an aeroponic system within a tropical
greenhouse, with the manipulation of only RZTs He et al. (2013)
Frontiers in Plant Science | www.frontiersin.org
and He (2014) and in the absence of any hormonal applications.
Therefore, this study describes a rapid and efficient rooting and
whole plant regeneration methodology for six Brassica species.
This method of root and shoot development using aeroponics can
be applicable to all commercial Brassica cultivars. The findings of
this study could be applied in the mass propagation of vegetable
crops, shortening the growth cycle as seed germination and
seedling development periods can be eliminated.
MATERIALS AND METHODS
Plant Material and Growth Conditions
The six cultivars of Brassica spp used in the study were: Kai
Lan (B. alboglabra), purple curly kale (B. oleracea var. acephala),
Mizuna (B. rapa var. nipposinica), Pai Tsai, Nai Bai C, and Nai
Bai T (B. rapa L. ssp. chinensis). The seeds were germinated
at 25◦ C room temperature on wet tissue paper in petri plates
for 3 days. Seedlings were inserted into polyurethane cubes
and acclimatized at 35◦ C in the greenhouse for 3 days before
transplanting into the aeroponic troughs at 20 ± 2◦ C cool root
zone temperature (C-RZT) and 30 ± 2◦ C ambient root zone
temperature (A-RZT), respectively. The roots of all plants were
misted with full strength (pH 6.8, EC 2.2 mS) Netherlands
Standard Composition (Douglas, 1982) nutrient solution for
1 min at 5 min intervals. All these conditions of aeroponic
troughs were maintained constant throughout the experiment.
Midday shoot temperatures varied between 35 to 40◦ C. After
45 days of transplanting, at the time of harvesting stage, few
of the stem segment explants with one leaf were transplanted
in their respective A- and C-RZTs to raise second generation
vegetables. Another set of the seeds was germinated provided
with a same nutrient solution, on the same day of explants
transplantation as controls to compare the growth performance
between explants and controls. Explants started rooting and
established few short roots at the time of control seedling
establishment in both RZTs. Hence, the establishment time
period (1 week) is a prerequisite for both explants and control
seeds for rooting and seed germination/seedling development,
respectively. However, explants showed vigorous growth as
compared to control seedlings in later stages of development.
Plant Histology
Explants of all six varieties of Brassica species followed
same rooting orientation in a circular mode toward the
parenchymatous cortex region (Figure 1). Therefore, as an
example tender and small stems of Kai Lan explant samples were
collected during the callus formation and root initiation and fixed
them in 10% formaldehyde for 3 days before being dehydrated
through graded solutions of alcohol, cleared in xylene, and
infiltrated with wax. Embedded sections were transversely and
longitudinally sectioned using Leica rotary microtome at 7 µm
thicknesses and transferred onto glass slides (after spreading
well in a water bath at 45◦ C). The slides were dried on the
hot plate at 45◦ C for 5 min. Completely dried and water free
slides were dipped twice in xylene for 5 min each, twice in 100%
ethanol for 5 min, once in 95% ethanol for 1 min and again
2
June 2016 | Volume 7 | Article 943
Srikanth et al.
Brassica Root Regeneration in Aeroponics
Measurements of Photosynthetic
Parameters
Newly expanded leaves of intact plants were analyzed for
photosynthetic parameters such as light saturated photosynthetic
CO2 assimilation rate (Asat ), stomatal conductance (gssat ),
intercellular CO2 concentration (Ci ) and transpiration rate using
an open infrared gas analysis system (LI-COR), 6 weeks after
the establishment of both explants and controls in C-RZT and
A-RZT. The LED light source was set at a photosynthetic photon
flux density of 1000 µmol m−2 s−1 . Leaf chamber temperature,
relative humidity, and average ambient CO2 concentration were
29◦ C, 70% and 396 ± 3 µmol mol−1 , respectively.
Measurements of Growth, Productivity,
and Water Content
Matured explants and controls were harvested 45 days after
transplant and germination, respectively, to obtain shoot and
root fresh weights (FWs) and dry weights (DWs). For FW, the
roots of plants were blotted with a tissue to remove excess water
prior to weighing. DW was determined after drying the same
plant samples at 100◦ C for 72 h. The means for each cultivar
was determined from three plants. Morphological traits, such as
leaf area, and shoot and root lengths, were also photographed
and measured. Leaf area was measured using WinDIAS3 v3.2.1
(Delta-T Devices Ltd). Water content (WC) was calculated as
follows: WC = [(FW−DW)/FW] × 100%.
RESULTS
The Efficiency of Different Brassica
Vegetable Explants to Generate Roots
Stem cuttings/explants of 20 for each mode of cutting from
lower, middle, and top portions (Figure 2A) of the donor plants
were tested for the rooting ability in both RZTs to avoid highest
mortality rate in explants propagation (Table 1). During this
investigation, the highest rooting ability was found in all explants
with shoot apical meristem (SAM) that grew under C-RZTwhich
can be attributed to the coordination between SAM and root
development in C-RZT. All explants rooted in the same level
under A-RZT except Mizuna explants which died due to exposure
to the highest midday light intensity up to 1000 µmolm−2 s−1 and
air temperature ≤40◦ C. KaiLan, Nai Bai C, and Nai Bai T explants
from the middle and lower portions of their respective donor
plants were able to root normally and developed new shoots from
axillary meristems. Whereas, Pai Tsai and kale explants from
middle and lower portions were unable to generate new roots
and shoots and eventually died after 2–3 days of planting on both
C-RZTand A-RZT.
FIGURE 1 | The comparable orientation of adventitious rooting in a
circular form toward the parenchymatous cortex in the cut ends of six
Brassica sp explants. (A) Kai Lan. (B) Purple curly kale. (C) Mizuna. (D) Pai
Tsai. (E) Nai Bai C. (F) Nai Bai T.
once in 70% ethanol for 1min. The slides were then air dried
and stained in 0.05% (w/v) toluidine blue in distilled water for
2 min, rinsed three times with deionized water to remove excess
stain. The slides were then dried and mounted using PermountTM
mounting medium (ProSciTech, Thuringowa, Australia). Root
generation from stem segments of Kai Lan was examined by using
Olympus BX60 microscope.
Measurements of Leaf Photosynthetic
Pigments
Samples were collected from explants and controls after 6 weeks
of their establishment in both RZTs. 0.05 g of fresh leaves
of three plants of each variety were soaked in 5 ml N,
N-dimethylformamide and left in the dark at 4◦ C for 48 h.
Absorption at wavelengths of 480 nm, 647 nm, and 664 nm were
measured using a spectrometer (UV-2550, Shimadzu, Japan) and
concentrations of chlorophyll a, chlorophyll b and carotenoids
were then calculated (Wellburn, 1984).
Frontiers in Plant Science | www.frontiersin.org
De novo Adventitious Root Formation in
Aeroponics
Soon after planting the stem segment explants on 20 ± 2◦ C
and 30 ± 2◦ CRZTs in tropical Aeroponics, the outer layer of
wounded cells died and later formed a necrotic dark layer. This
layer helped to protect the cut surface from desiccation and
3
June 2016 | Volume 7 | Article 943
Srikanth et al.
Brassica Root Regeneration in Aeroponics
FIGURE 2 | Early events of callus formation and elongation of adventitious roots (ARs) in Kai Lan stem explants. (A) Kai Lan plants with cutting regions
and explants positions. (B) Arrow pointing to the callus initiation at the cut end. (C) Roots formed in a circular ring. (D) Root elongation. (E) Arrow showing the
longitudinal section of fresh regenerating stem showing degenerating central pith region.
pathogens attack. Living cells underneath this dark layer has
begun divisions after 2 days of wounding and an outer layer of
parenchyma cells after rapid mitotic divisions formed a circular
mass of cells which later formed little white undifferentiated
tissue called callus (Figure 2B). At the base of cut end,
anticlinal and periclinal divisions of the cambial cells resulted
in secondary xylem toward pith and secondary phloem toward
cortex (Figure 3). The xylem vessels became a thick walled to
ease the rapid hydraulic conductivity for regenerating explants
(Figures 3C,D).
The newly formed phloem parenchymatous cells after
vigorous cell divisions formed root callus growth proliferations
(Figures 4, 5, and 6A). Root initials formed from the callus
in the vicinity of the vascular cambium and phloem ray
parenchyma (Figure 6A), which had become meristematic
by dedifferentiation. Further cell divisions occurred and the
meristematic area had become more organized with the
formation of a root initial (Figures 2 and 6). Ultimately, these
root initials developed into an organized root primordia in
the secondary phloem and cortex (Figure 6A). Later, the root
primordia grew outwardly through stem tissues and formed
the vascular tissue connections between the primordia and
vascular tissue of stem cutting. Upon emergence from the
stem segment, the ARs have already developed a root cap as
well as a complete vascular connection with the originating
stem (Figures 6D–H). Eventually, parenchymatous cells of the
cortex also contributed to root formation covering the entire
base with many roots and root initials (Figures 5B and 6H).
Visible root initials emerged from the cuttings after 3–5 days
of planting in C-RZT aeroponics whereas roots were visible
only after 4–7 days of planting inA-RZT. Once primordia
are formed, there was a comparable time period of 5–7 days
between root primordia elongation (emergence) and maximum
rooting in both RZTs. Even in the C-RZT, some of the
TABLE 1 | Stem segment ex-plant rooting ability in six Brassica species
B. alboglabra (KaiLan), B. oleracea var. acephala (purple kale), B. rapa L.
ssp. Chinensis L (Pai Tsai, Nai Bai C, and Nai Bai T).
Cultivar name
Lower stem with
root system
Middle stem
cuttings
Shoot apical
meristem
Kai Lan
X
X
X
Mizuna
X
–
X
Kale
7
7
X
Pai Tsai
7
7
X
Nai Bai C
X
X
X
Nai Bai T
X
X
X
Frontiers in Plant Science | www.frontiersin.org
4
June 2016 | Volume 7 | Article 943
Srikanth et al.
Brassica Root Regeneration in Aeroponics
FIGURE 3 | Anatomy of AR formation from phloem parenchyma in Kai Lan stem explants of control (A,B-arrows showing normal tissues) and rooted
(C,D-arrows showing the abnormal tissues and cell division) samples. (A) Control stem transverse section showing epidermis (ep), cortex parenchyma (cp)
and pith (pi) in normal tissue orientation. (B) Control stem showing vascular bundle (phloem (ph), cambium (ca), metaxylem (mx), protoxylem (px), xylem parenchyma
(xp), and pith (pi) with aregular arrangement of cells. (C) Rooted stem transverse section showing irregular epidermis (ep) and cortex parenchyma (cp) with an
abnormal tissue orientation. (D) The continuing mitotic activity of phloem parenchyma cells led to a ring of conspicuous meristematic tissue complexes evident in the
cortex region of the rooted stem section. Vascular bundle showing the secondary xylem (sx) and phloem (ph) cells derived from active cambial cells (ca).
as compared to other three vegetable explants such as Kale, Pai
Tsai, and Mizuna. In C-RZT, Kai Lan explants exhibited 95%
survival rate among all the Kai Lan cuttings planted, whereas
explants in A-RZT showed 80% survival rate and 20% mortality
rate. However, Nai Bai C and Nai Bai T explants showed the
highest survival rate as 96 and 98% in C-RZT and 90 and 93%
survival rate in A-RZT, respectively. Both Mizuna and Kale
explants showed 60% survival rate in C-RZT and only Kale
explants survived up to 45% in A-RZT. While the survival rate
of Pai Tsai explants was 75% in C-RZT and 70% in A-RZT. In
C-RZTs, control seedlings from six vegetable varieties established
purple kale cuttings were delayed rooting even after 7 days
of planting. This delay was due to variability in cuttings from
different-sized stock plants but once root primordia formed, root
emergence consistently occurred within a week period in both
RZTs.
Explant Establishment, Growth, and
Productivity
Among six vegetable explants planted on C- and A-RZTs, Kai
Lan, Nai Bai C, and Nai Bai T showed the highest survival rate
Frontiers in Plant Science | www.frontiersin.org
5
June 2016 | Volume 7 | Article 943
Srikanth et al.
Brassica Root Regeneration in Aeroponics
FIGURE 4 | Cell divisions in cortex and deterioration in pith occurred post root formation from the secondary phloem. (A) Transverse section of Kai Lan
control stem showing normal epidermis (ep) and cortex parenchyma (cp) regions. (B) Control stem showing normal pith at the center. (C) Transverse section of
rooted stem showing active mitotic divisions in the cortex to initiate meristematic region. (D) Rapid mitotic divisions resulted in a circular mass of tissue in cortex
parenchyma (cp). (E) Rooted stem showing early events of deterioration in central pith (pi). (F) Enlarged picture showing the cell disintegration in central pith (pi)
(arrows pointing toward the cell division).
leaf area in A-RZT (Figure 7). Although there was not much
difference in shoot length of Mizuna explants and controls, it
was evident from the results that Mizuna explants grew well
with more leaves in the C-RZT and showed almost threefold
increased leaf area. Whereas kale, Pai Tsai, Nai Bai C, and Nai
Bai T explants showed low to moderate differences in the plant
heights and leaf area measurements, but still explants of C-RZT
were significantly (∗ p < 0.05, ∗∗ p < 0.01) superior to the A-RZT
(Table 2, Figure 7).
Total fresh and DWs of shoots and roots were determined
from a single harvest of three plants from each variety when
explants reached harvest maturity. In correlation with the
plant stature, the biomass (FW and DW) of all vegetable
explants grown under cool and A- RZTs showed the highest
and remarkable increase (∗ p < 0.05, ∗∗ p < 0.01) compared
to the control plants (Figure 8). However, FW and DW were
significantly affected by imposing different RZTs. Among all
six vegetable varieties, Kai Lan explants from C-RZT showed
almost twofold increased FW in concurrence with their large
plant stature. However, from the data, it was evident that the DW
of all vegetables decreased drastically as they possessed a high
successfully, exhibiting up to 98% survival rate in Kai Lan,
Nai Bai C, and Nai Bai T, 95% in Mizuna and Kale, whereas
96% in Pai Tsai. But in A-RZT, all six varieties showed 1 to
2% less establishment rate as compared to C-RTZ. Since all
explants/cuttings used in this experiment were almost uniform in
each species, the number of growth measurements such as shoot
length, root length, leaf area, fresh and DWs of shoots and roots
were recorded and analyzed.
Application of different RZTs significantly affected the plant
growth components, especially shoot and root lengths. Explants
grown in both RZTs showed the highest biomass as compared
to their controls. Nevertheless, explants of C-RZT were large
stature with increased biomass compared to the explants of
A-RZT. While this difference in the plant stature and biomass
was also observed in the control plants of both RZTs. Moreover,
all results indicating clearly that the controls were significantly
smaller with very low biomass when compared with the explants
in both RZTs (Table 2; Figures 7 and 8). Kai Lan explants
showed almost 12.4 cm shoot length and 23.5 cm root length
difference from C- to A-RZTs (Table 2). This larger difference in
Kai Lan plant stature was also correlated with twofold decreased
Frontiers in Plant Science | www.frontiersin.org
6
June 2016 | Volume 7 | Article 943
Srikanth et al.
Brassica Root Regeneration in Aeroponics
amount of water content in the root and shoots. Explants of Kai
Lan shoots and roots showed 93% WC in the C-RZT whereas
92% in A-RZT. Mizuna explants possessed almost 91% WC in
both roots and shoots, whereas controls showed 92% WC in both
RZTs. Kale explants showed almost 90% WC in shoots and roots
in both RZTs. While shoots of kale controls contained with 90.5%
in C-RZT and 87% in A-RZT. But kale control roots retained
92.8% WC in A-RZT and only 84% in C-RZT. Whereas, all Pai
Tsai, NaiBai C, and NaiBai T plants (both explants and controls)
showed 95% WC in shoots and up to 91% WC in roots of both
RZTs. From the results, it was clear that almost all plants retained
more than 90% WC.
Photosynthetic Pigments and
Performance
Kai Lan explants had lower carotenoid content (Figure 9C)
in C-RZT compared to A-RZT. In C-RZT, Mizuna explants
had higher chlorophyll a/b ratio (Figure 9A), total chlorophyll
(Figure 9B) and carotenoid content than its control plants. For
Nai Bai C, there was higher total chlorophyll and carotenoid
content in C-RZT for the control plants, but the reverse
was observed for its explants. Such similar results were also
observed in Nai Bai T, with its chlorophyll/ carotenoid ratio
(Figure 9D) being lower for both control and explant. However,
the chlorophyll/carotenoid was similar for both control and
explants of Nai Bai C. Asat (Figure 10A) was higher for all Kai
Lan plants in C-RZT, but similar between control and explants in
both RZTs for the rest of the plant types. gssat (Figure 10B) was
generally higher for most plants in C-RZT with the exception of
FIGURE 5 | Longitudinal sections of Kai Lan control and rooted stems
showing variation in tissue orientation. (A) Longitudinal section of the
control stems (arrow indicating the blunt end without any divisions).
(B) Longitudinal section of rooted stem showing rooting from basal wound
tissue (arrow indicating the cell divisions at the cut end). (C) Enlarged picture
of the control stem longitudinal section showing epidermis (ep), cortex
parenchyma (cp), the arrow showing the regular phloem (ph), cambium (ca)
xylem (x) and pith (pi). (D) Enlarged picture of rooted stem L.S showing
epidermis (ep), cortex parenchyma (cp), pith (pi) and an arrow showing the
irregular vascular bundles with rapidly dividing cells.
FIGURE 6 | Histological characteristics of AR development in Kai Lan stem explants. (A–D) Transverse sections showing early events of mitotic divisions
and callus initiation. (A) Vascular bundle showing thick-walled vascular tissues (Arrow pointing to the callus initiation from phloem parenchyma). (B) Arrows showing
the mitotic cell divisions. (C) Arrows indicating the callus initials. (D) Arrow showing the round callus. (E–H) Arrows in the longitudinal sections showing root
primordial initiation from callus (E,F), root cap development (G) and AR formation from meristematic region of explant cut ends (H).
Frontiers in Plant Science | www.frontiersin.org
7
June 2016 | Volume 7 | Article 943
Srikanth et al.
Brassica Root Regeneration in Aeroponics
TABLE 2 | Explants and controls showed a significant difference (∗ p < 0.05, ∗∗ p < 0.01; n = 10) in their shoot and root length under C- and A-RZTs.
Plant sample
C-RZT
Shoot length (cm)
A-RZT
Root length (cm)
Shoot length (cm)
Root length (cm)
Kai Lan explant
45.5 ± 1.3∗∗
74.8 ± 3.6∗
33.1 ± 2.4∗
51.3 ± 1.2∗∗
Kai Lan control
24.6 ± 0.6
51.3 ± 4.3
22.7 ± 2.6
43.6 ± 1.0
Mizuna explant
34.0 ± 2.6
83.3 ± 4.7∗∗
–
–
Mizuna control
30.0 ± 0.5
59.5 ± 1.3
23.9 ± 1.6
28.3 ± 3.8
Kale explant
37.4 ± 2.8∗∗
69.0 ± 4.7∗∗
25.9 ± 4.2∗
41.0 ± 1.0
Kale control
16.0 ± 1.7
38.3 ± 0.6
12.0 ± 0.5
36.8 ± 3.5
Pai Tsai explant
27.0 ± 1.1
80.0 ± 6.2∗
23.3 ± 2.6
45 ± 1.5
Pai Tsai control
22.0 ± 1.6
51.3 ± 4.4
22.6 ± 1.5
38.3 ± 4.4
Nai Bai C explant
15.5 ± 0.8
61.3 ± 3.6
16.3 ± 2.7
59.6 ± 4.3∗
Nai Bai C control
11.1 ± 1.6
55.0 ± 5.5
10.3 ± 0.3
29.6 ± 3.7
Nai Bai T explant
18.5 ± 1.8
69.1 ± 6.7∗∗
14.5 ± 1.0∗
Nai Bai T control
15.1 ± 0.6
42.0 ± 3.7
10.5 ± 0.5
stimulus, such as wounding (Abarca and Díaz-Sala, 2009).
Complex cellular processes involved in the AR formation are cell
reorganization, induction of cell divisions, the organization of a
root primordium and root development and emergence (Legué
et al., 2014).
The AR formation was direct; i.e., in an organized mode
without a long intervening period of callus formation. The roots
grew through the cortex and often emerged out from a small
round callus during the 3 to 7 day’s period after inserting the
cuttings into aeroponic boards (in both RZTs). Interestingly,
before root emergence, we have observed a ring of secondary
vascular tissues developed from the actively dividing cambial
cells at the wound site. During the rooting, this kind of tissue
differentiation was observed only at the cut end and not above
the wound region. Hence, it is emphasized that the wound
signaling and continuous nutrient solution mist had led to the
immediate auxin accumulation at the cut end which induced
few small calluses and then rooting. At the base of cut end,
anticlinal and periclinal divisions of the cambial cells resulted
in secondary xylem toward pith and secondary phloem toward
cortex. The continuing mitotic activity of secondary phloem
parenchyma cells led to a ring of conspicuous white meristematic
tissue complexes called ‘callus’. Root initials formed from the
callus which in turn developed into ARs in the vicinity of
the vascular cambium and phloem ray parenchyma. The study
highlights that the hormone free cuttings can produce roots
at multiple positions around the vascular tissue and so this
propagation method can produce more ARs at the base of each
cutting which resulted in higher survival and growth rate of
explants.
In this experiment, significant results have been recorded for
stem cutting propagation which would be demonstrating the
possibility and success rate of vegetative propagation in tested
Brassica samples and other vegetable species. The results are
also representing the significant difference in the biomass of
explants and controls. Among all, Kai Lan explants showed
more remarkable FW and DW in cool RZT compared with
other explants and controls. Since, explants were collected from
the matured harvest stage donor plants, their well-established
FIGURE 7 | Total leaf area of explants and controls of six cultivar
varieties of Brassica spp. grown under C-RZT (20 ± 2◦ C) and A-RZT t
(30 ± 2◦ C) in a tropical aeroponics greenhouse for 45 days. Each bar
graph is the mean of 3 measurements from three different plants (n ≥ 3).
Vertical bars represent standard errors. Significance: ∗∗ p < 0.01.
Kai Lan which had lower gssat . Transpiration rate (Figure 10D)
was generally lower in C-RZT, than A-RZT, for all plants except
for Nai Bai C which had similar rates at both RZTs. Nai Bai
T had higher gssat (Figure 10B) and Ci (Figure 10C), though
a significantly lower transpiration rate for both control and
explants, at C-RZT.
DISCUSSION
Plant tissues have the enormous regeneration capacity, and
entire plant can be developed from a single cell or small
cuttings/explants (Xu and Huang, 2014). The AR formation
is of great importance for vegetative propagation, but difficult
to achieve in many crop species. In the present study, ARs
were successfully developed from stem-segment explants of
Brassica species on tropical aeroponics. De novo induction of
roots from stem cuttings in plants involve the induction of
meristems from adult somatic cells that are not determined
to originate a meristem in normal development. Usually, AR
formation is induced in stem cuttings, which experience a
Frontiers in Plant Science | www.frontiersin.org
51 ± 4.5
36.6 ± 3.6
8
June 2016 | Volume 7 | Article 943
Srikanth et al.
Brassica Root Regeneration in Aeroponics
FIGURE 8 | Fresh and dry weights (DWs) of shoots and roots of explant and control plants. Six cultivar varieties of Brassica spp. grown under C-RZT
(20 ± 2◦ C) and A-RZT (30 ± 2◦ C) in a tropical aeroponics greenhouse for 45 days. Each bar graph is the mean of three measurements from three different plants
(n ≥ 3). Vertical bars represent standard errors. Significance: ∗ p < 0.05, ∗∗ p < 0.01.
FIGURE 9 | Chlorophyll a/b ratio (A), total chlorophyll concentration (B), carotenoid concentration (C), and chlorophyll/ carotenoid ratio (D) of Kai
Lan, Mizuna, Nai Bai C, and Nai Bai T grown in A-RZT and C-RZT. Each bar graph is the mean of three measurements from three different plants (n ≥ 3).
Vertical bars represent standard errors. Significance: ∗ p < 0.05, ∗∗ p < 0.01.
Frontiers in Plant Science | www.frontiersin.org
9
June 2016 | Volume 7 | Article 943
Srikanth et al.
Brassica Root Regeneration in Aeroponics
FIGURE 10 | Photosynthetic parameters such as light saturated photosynthetic CO2 assimilation rate (Asat ) (A), stomatal conductance (gssat ) (B),
intercellular CO2 concentration (Ci ) (C), and transpiration rate (D) of Kai Lan, Mizuna, Nai Bai C, and Nai Bai T grown in A-RZT and C-RZT. Each bar
graph is the mean of three measurements from three different plants (n ≥ 3). Vertical bars represent standard errors. Significance: ∗ p < 0.05, ∗∗ p < 0.01. Amb:
ambient temperature; Cool: cool temperature.
biomass content, they encountered a poor establishment problem
in A-RZT. While other vegetables such as Pai Tsai, Nai Bai C,
and Nai Bai T had shown greater establishment rate and yielded
more in both RZTs. C-RZT also promoted the higher shoot
and root lengths of Kai Lan, Mizuna, Kale, Pai Tsai, Nai Bai C,
and Nai Bai T explants, suggested that plants grown in C-RZT
possess a higher water and nutrition uptake capacity, which
would contribute to the productivity of plants. The ratio of leaf
chlorophyll a and chlorophyll b contents were not significantly
different among controls and explants in both RZTs. However,
total chlorophyll content is slightly higher in most of the plants
in A-RZT. Nevertheless, total chlorophyll/ carotenoid ratio found
to be almost similar in controls and explants of both RZT,
except Nai Bai which showed a little higher ratio in A-RZT.
These results demonstrating that the photosynthetic pigments
contents are species specific and their performance is unique in
each species. Photosynthetic parameters such as light saturated
photosynthetic CO2 assimilation rate (Asat ) is considerably
higher in almost all explants at both RZTs, while stomatal
conductance (gssat ) is higher in C-RZT plants. Inter cellular CO2
concentration (Ci ) is not much influenced by different RZTs and
is relatively uniform among all plants. Whereas, transpiration
rate is significantly higher in A-RZT plants compared to C-RZT
plants except Nai Bai. Despite all variations in photosynthetic
parameters, all explants were healthy and well advanced in
growth in comparison to control plants. The above discussion is
meristematic regions, and continuous nutrient supply through
root zone area of aeroponics could be contributed for their quick
recovery and root formation in a few days’ period. Once explants
established the root system, they started growing vigorously and
resulted in large plant stature. Meanwhile, seeds germinated
on the same day of the explants planting took 2–3 days for
germination and then 3 days for the establishment. But even
after transplanting on the aeroponic systems, seedlings grew
very slowly in the first few days until they established potential
meristematic regions and root systems to support rapid growth.
Eventually, seedlings of all vegetable varieties (Kai Lan, Mizuna,
Kale, Pai Tsai, Nai Bai C, and Nai Bai T) started growing
rapidly after one month of transplanting. While control plants
reached halfway to the harvest stage, explants were grown up
to harvest stage. This resulted in the high biomass content (FW
and DW) of the roots and shoots of explants compared to
control plants. While total leaf area was influenced by C-RZTs
conditions, as there were more leaf number and specific leaf
area was recorded little higher than A-RZT. However, explants
recorded highest SLA compared to control plants due to changes
in leaf thickness and leaf density. This simple and reliable
method of explants regenerate into a whole plant, thereby rapid
vegetative propagation is suitable for the vegetable varieties tested
in both RZTs except Mizuna in A-RZT. But still, Mizuna showed
significantly increased biomass when compared to its control
plants in C-RZT. Even though Kale explants showed higher
Frontiers in Plant Science | www.frontiersin.org
10
June 2016 | Volume 7 | Article 943
Srikanth et al.
Brassica Root Regeneration in Aeroponics
also demonstrating that the effect of C-RZT and A-RZT is again
species-dependent.
In the present study, by using the aeroponic systems we just
studied the AR formation and successful establishment of the
stem segment explants of Brassica vegetable species without using
any plant hormones, which would be useful in the vegetative
propagation of leafy vegetable crops that has huge demand
worldwide. Moreover, there is a need to develop different farming
systems to secure the continuous vegetable production in space
limited cities such as Singapore. By adapting new cultivating
techniques like vegetative propagation on aeroponic farming
systems, constant leafy vegetable supply is conceivable and
compels the vegetable import in urban areas. There may be other
instances in which aeroponic vegetative propagation can be used
as an alternative to seed propagation. They include easy and rapid
multiplication of selected genotypes which are generated from the
conventional breeding program or induced variants from cells,
tissue, or organ culture, genetic transformation, propagation
of parents for hybrid seed production and speedy propagation
of asexually propagated crops. Aeroponic propagation is also
feasible for controlling pollination techniques such as cross
pollination, self-pollination or hand pollination in the hybrid
breeding program. Moreover, this method of hormone free AR
formation and clonal propagation is useful for woody species that
are often vegetatively propagated by stem cuttings.
REFERENCES
Rochfort, S. J., Imsic, M., Jones, R., Trenerry, V. C., and Tomkins, B. (2006).
Characterisation of flavonol conjugates in immature leaves of Pak Choi
(Brassica rapa L). Ssp chinensis L. (Hanelt) by HPLC-DAD and LC-MS/MS.
J. Agric. Food Chem. 54, 4855–4860.
Velasco, P., Cartea, M. E., Gonzalez, C., Vilar, M., and Ordas, A. (2007). Factors
affecting the glucosinolate content of kale (Brassica oleracea acephala group).
J. Agric. Food Chem. 55, 955–962. doi: 10.1021/jf0624897
Weathers, P. J., and Giles, K. L. (1988). Regeneration of plants using nutrient mist
culture. In Vitro Cell. Dev. Biol. Anim. 24, 727–732.
Welander, M., Geier, T., Smolka, A., Ahlman, A., Fan, J., and Zhu, L. H.
(2014). Origin, timing, and gene expression profile of adventitious
rooting in Arabidopsis hypocotyls and stems. Am. J. Bot. 101, 1–12. doi:
10.3732/ajb.1300258
Wellburn, A. R. (1984). The spectral determination of chlorophylls A and B,
as well as total Carotenoids. using various solvents with spectrophotometers
of different resolution. J. Plant Phys. 144, 307–313. doi: 10.1007/s11120-0139834-1
Xu, L., and Huang, H. (2014). Genetic and epigenetic controls of plant
regeneration. Curr. Top. Dev. Biol. 108, 1–33. doi: 10.1016/B978-0-12-3914989.00009-7
Yang, Q., Chauvin, J. E., and Herve, Y. (1992). A study of factors affecting anther
culture of cauliflower (Brassica oleracea var. botrytis). Plant Cell Tiss. Org. Cult.
28, 289–296. doi: 10.1007/BF00223783
Zhao, J. (2007). The Genetics of Phytate Content and Morphological Traits in
Brassica Rapa. Ph.D. thesis, Wageningen University, Wageningen.
Zobel, R. W. (1989). “Steady-state control and investigation of root system
morphology,” in Steady State and Continuous Control of Root Growth, eds J. G.
Torrey and L. Winship (Amsterdam: Kluwer Academic Publishers), 165–182.
AUTHOR CONTRIBUTIONS
ZC initiated the project. SS and TWC performed experiments. AY
contributed to aeroponics plant care. SS, TWC, JH, and ZC wrote
the manuscript.
FUNDING
NIE AcRF grant (RI 3/13 CZ) and MOE Tier 1 grant (RP 1/14
CZ).
ACKNOWLEDGMENTS
The project was funded by NIE AcRF grant (RI 3/13 CZ) and
MOE Tier 1 grant (RP 1/14 CZ). The authors wish to thank Dr.
Kin Wai Lai from Singapore Polytechnic for providing necessary
facilities for microtome sectioning in the course of work.
Abarca, D., and Díaz-Sala, C. (2009). “Adventitious root formation in conifers,” in
Adventitious root formation of forest trees and horticultural plants–from genes
to applications, eds K. Niemiand and C. Scagel (Kerala: Research Signpost
Publishers), 227–257.
Cheng, F., Liu, S., Wu, J., Fang, L., Sun, S., Liu, B., et al. (2011). BRAD, the
genetics and genomics database for Brassica plants. BMC Plant. Biol. 11:136.
doi: 10.1186/1471-2229-11-136
Douglas, J. S. (1982). Advanced Guide to Hydroponics. Dehra Dun: Natraj
Publishers.
Ellis, R. (2007). “The Encyclopedia of Seeds,” in Science Technology and Uses,
eds M. Black, J. D. Bewley, and P. Halmer (Wallingford,CT: CABI), 900.
ISBN-9780851997230.
Fink, S. (1999). “Root regeneration” in Pathological and Regenerative Plant
Anatomy. Encyclopedia of plant anatomy, ed. S. Fink (Gebr.Bornträger: Berlin),
6, 350–371.
He, J. (2014). “Elevated root-zone CO2 on photosynthesis of vegetable crops
at different air and root-zone temperatures,” in Photosynthesis: Functional
Genomics, Physiological Processes and Environmental Issues, ed. N. Khan
(New York, NY: Nova Science Publishers), 23–54.
He, J., Qin, L., and Lee, S. K. (2013). Root-zone CO2 and root-zone temperature
effects on photosynthesis and nitrogen metabolism of aeroponically grown
lettuce (Lactuca sativa L.) in the tropics. Photosynthetica 50, 330–340. doi:
10.1007/s11099-013-0030-5
Legué, V., Rigald, A., and Bhalerao, R. P. (2014). Adventitious root formation in
tree species: involvement of transcription factors. Physiol. Plant. 151, 192–198.
doi: 10.1111/ppl.12197
Luo, H. Y., He, J., and Lee, S. K. (2012). Interaction between potassium (K)
concentration and root-zone temperature (RZT) on growth and photosynthesis
of temperate lettuce grown in the tropics. J. Plant Nutr. 35, 1004–1021. doi:
10.1080/01904167.2012.671404
Parkin, I. A. P., Koh, C., Tang, H. B., Robinson, S. J., Kagale, S., Clarke, W. E.,
et al. (2014). Transcriptome and methylome profiling reveals relics of genome
dominance in the mesopolyploid Brassica oleracea. Genome Biol. 15, R77. doi:
10.1186/gb-2014-15-6-r77
Rakow, G. (2004). “Species origin and economic importance of Brassica,” in
Biotechnology in Agriculture and Forestry, eds E. C. Pua and C. J. Douglas
(New York, NY: Springer-Verlag Berlin Heidelberg), 54, 3–11.
Frontiers in Plant Science | www.frontiersin.org
Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2016 Srikanth, Choong, Yan, He and Chen. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other forums is permitted, provided the
original author(s) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use, distribution
or reproduction is permitted which does not comply with these terms.
11
June 2016 | Volume 7 | Article 943